recombinant mouse angpt2 protein  (R&D Systems)

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Effects of Dual Inhibition of VEGF-A and Angpt-2 on Angiogenesis and Lymphangiogenesis in an Alkali-Induced Corneal Injury Model.

doi: 10.1111/gtc.70035

Figure Lengend Snippet: FIGURE 2 | Neutralizing antibodies anti-VEGFA and/or ANGPT2 suppressed angiogenesis but not lymphangiogenesis in the alkali treated mouse cornea. Mice were anesthetized, and the corneal thickness was measured using the B-scans from AS-OCT. Then, the cornea was treated with alkali, and the antibodies were administered. After 10 days, mice were anesthetized, and the corneal thickness was measured before harvest. Flat- mount immunostaining using anti-CD31 and -LYVE1 antibodies was performed. The whole area was divided into three parts (CD31) or two parts (LYVE1), and immunostained signals were quantified using AngioTool as described in the Methods section. (A–C) CD31 signals of vessel areas of the outer, middle, and inner areas are shown. (D, E) LYVE1 signals of vessel areas of the outer and inner areas are shown. Values represent the averages of 10 to 14 independent samples with their standard deviations. (F) Thickness of the cornea. Values are for the alkali-treated Day 10 samples relative to the value before treatment in the same eye. Values represent the averages of nine independent samples with their standard deviations. Statistical analysis was performed using one-way ANOVA followed by Tukey's HSD multiple-comparison test.

Article Snippet: Recombinant mouse ANGPT2 (50 ng, R&D systems, 7186- AN) and VEGF165 (50 ng, PeproTech, 450– 32) were also administered at the dorsal and ventral points by subconjunctival injection.

Techniques: Immunostaining, Comparison

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Effects of Dual Inhibition of VEGF-A and Angpt-2 on Angiogenesis and Lymphangiogenesis in an Alkali-Induced Corneal Injury Model.

doi: 10.1111/gtc.70035

Figure Lengend Snippet: FIGURE 3 | Gene expression patterns of cornea after alkali treatment in presence or absence of anti-VEGFA_ab, -ANGPT2_ab, or BsAb. Gene expression patterns of cornea after alkali treatment in the presence or absence of the antibodies. Primers for angiogenesis related cytokines (A), blood vessel (Pecam1), and lymphatic vessel (Pdpn) specific marker genes (B), and inflammatory cytokines (C) were used for qPCR. Corneas were harvested after 10 days of alkali treatment, and RT-qPCR was performed. Samples represent the average of 4 to 5 independent samples with their stan- dard deviations. Statistical analysis was performed using one-way ANOVA followed by Tukey's HSD multiple-comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Recombinant mouse ANGPT2 (50 ng, R&D systems, 7186- AN) and VEGF165 (50 ng, PeproTech, 450– 32) were also administered at the dorsal and ventral points by subconjunctival injection.

Techniques: Gene Expression, Marker, Quantitative RT-PCR, Comparison

Journal: eLife

Article Title: Angiopoietin-4-dependent venous maturation and fluid drainage in the peripheral retina

doi: 10.7554/elife.37776

Figure Lengend Snippet: Figure 3. mRNA expression of angiopoietins and Tie2 in retinal vein development. mRNA expression was detected in whole mount retinas from WT mice by in situ hybridization. (A) At P3, astrocytes expressing Angpt4 precede the front of the developing vasculature (red blood cells indicated by arrows). (B) At P12, Angpt4 expression is notable around the developing vein in the peripheral retina (V, asterisks) and colocalizes with the astrocyte marker GFAP (white overlay in insert). (C) Angpt1 expression is not detected at P12 by whole mount in situ hybridization in the peripheral retina. (D) Angpt2 is expressed in retinal neurons (arrowhead) in the intermediate retinal plexus. (E) Expression of Tie2 is detected in the endothelium of arteries (A), veins and capillaries at P12. OR, ora serrata. DOI: https://doi.org/10.7554/eLife.37776.008 The following figure supplement is available for figure 3:

Article Snippet: Purified recombinant human ANGPT1, ANGPT2, and ANGPT4 and mouse Angpt4 were from R & D Systems.

Techniques: Expressing, In Situ Hybridization, Marker

Journal: Molecular Therapy. Nucleic Acids

Article Title: Angpt2 Induces Mesangial Cell Apoptosis through the MicroRNA-33-5p-SOCS5 Loop in Diabetic Nephropathy

doi: 10.1016/j.omtn.2018.10.003

Figure Lengend Snippet: Serum Angiopoietin-2 Is Positively Correlated with Urinary Albumin-Creatinine Ratio in Diabetes Mellitus (A) Serum Angiopoietin-2 (Angpt2) was more elevated in db/db mice (n = 3) than in db/m mice (n = 3). (B) A positive correlation between urinary albumin-creatinine ratio (ACR) and serum Angpt2 was found in mice. (C) Serum Angpt2 was more elevated in type 2 diabetes mellitus (DM) patients (n = 63) than in healthy individuals (n = 34). (D) A positive correlation between urinary ACR and serum Angpt2 was also found in human participants. Magnetic Luminex Assay was used to assess serum Angpt2. Urine albumin was measured using an immunoturbidimetric assay, and urine creatinine was determined by the enzymatic method. The bar graph represents the mean ± SEM. *p < 0.05 by two-tailed Student’s t test. p value of correlation by Spearman analysis.

Article Snippet: Recombinant mouse Angpt2 proteins (7186-AN), anti-Mouse/Rat Tie2 antibody (AF762), and control polyclonal Goat immunoglobulin G (IgG) (AB-108-C) were purchased from R&D Systems (USA).

Techniques: Luminex, Immunoturbidimetry Assay, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Angpt2 Induces Mesangial Cell Apoptosis through the MicroRNA-33-5p-SOCS5 Loop in Diabetic Nephropathy

doi: 10.1016/j.omtn.2018.10.003

Figure Lengend Snippet: Angpt2 Exhibits a Synergistic Effect on the Induction of Apoptosis in Mouse Mesangial Cells through Tie-2 Receptor under a High Glucose Condition (A) The effect of Angpt2 on cell viability of mouse mesangial cells (MMCs). MMCs were incubated for 48 hr with or without Angpt2 (300 ng/mL) under normal glucose (NG, 5.5 mM) or high glucose (HG) (25 mM) condition. Cell viability was assessed by WST-1 assay. (B) Angpt2 enhanced cell death under an HG condition in MMCs. MMCs were double stained with Annexin-V fluorescein isothiocyanate and PI and analyzed by flow cytometry. Cell apoptosis was expressed as the percentage of cells with Annexin-V and PI double staining in total cell populations. (C and D) Angpt2 aggravated caspase-3 (C) and caspase-9 (D) activation in MMCs treated with HG for 48 hr. (E and F) Angpt2 reduced B cell lymphoma 2 (Bcl-2) (E) and B cell lymphoma-extra large (Bcl-XL) (F) protein expression in MMCs under HG for 36 hr. The activities of caspase-3 and caspase-9 were detected by fluorescent caspase assay kits. Bcl-2 and Bcl-XL protein levels were assessed by western blotting. (G) Tie2 receptor involves Angpt2-mediated apoptosis in MMCs under an HG condition. MMCs were pre-incubated either with Tie2 antibody (5 μg/mL) or IgG (5 μg/mL) for 2 hr and treated with Angpt2 for another 48 hr under an HG condition. The bar graph represents the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by ANOVA followed by the post hoc test adjusted with a Tukey correction.

Article Snippet: Recombinant mouse Angpt2 proteins (7186-AN), anti-Mouse/Rat Tie2 antibody (AF762), and control polyclonal Goat immunoglobulin G (IgG) (AB-108-C) were purchased from R&D Systems (USA).

Techniques: Incubation, WST-1 Assay, Staining, Flow Cytometry, Double Staining, Activation Assay, Expressing, Caspase Assay, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: Angpt2 Induces Mesangial Cell Apoptosis through the MicroRNA-33-5p-SOCS5 Loop in Diabetic Nephropathy

doi: 10.1016/j.omtn.2018.10.003

Figure Lengend Snippet: Identification of Potential miRNAs Contributing to Angpt2-Mediated Apoptosis in MMCs under an HG Condition (A) The flowchart of miRNA identification and selection. (B) The heatmap revealed differentially expressed miRNAs from MMCs treated with HG + Angpt2 or HG with Z score values. The Z score of each miRNA is its expression level’s relationship to the mean of expression levels of all miRNAs. (C) Ingenuity Pathway Analysis (IPA) of miR-33-5p profiles of Angpt2-treated MMCs under an HG condition. (D) miR-33-5p levels in MMCs. MMCs were treated with or without Angpt2 under an NG or HG condition for 12 hr. miR-33-5p levels were assessed by real-time qPCR. The bar graph represents the mean ± SEM of at least three independent experiments. *p < 0.05 and ***p < 0.001 by ANOVA followed by the post hoc test adjusted with a Tukey correction.

Article Snippet: Recombinant mouse Angpt2 proteins (7186-AN), anti-Mouse/Rat Tie2 antibody (AF762), and control polyclonal Goat immunoglobulin G (IgG) (AB-108-C) were purchased from R&D Systems (USA).

Techniques: Selection, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Angpt2 Induces Mesangial Cell Apoptosis through the MicroRNA-33-5p-SOCS5 Loop in Diabetic Nephropathy

doi: 10.1016/j.omtn.2018.10.003

Figure Lengend Snippet: Angpt2-Induced MMC Apoptosis Is Mediated by miR-33-5p Inhibition under an HG Condition (A) miR-33-5p mimics prevent the synergistic effect of Angpt2 on apoptosis induction in MMCs under an HG condition. MMCs were transfected with either miR-33-5p mimic (200 nM) or control of mimic (miR-NC, 200 nM) for 24 hr and treated with Angpt2 (300 ng/mL) under an NG or HG condition for 24 hr. Apoptotic cells were assessed by flow cytometry with Annexin-V/PI double staining. (B) miR-33-5p inhibitor mimicked the effect of Angpt2 on apoptosis induction in MMCs under an HG condition. MMCs were transfected with miR-33-5p inhibitor (50 nM) or control of inhibitor (anti-miR-NC, 50 nM) for 24 hr and then incubated under an NG or HG condition for 24 hr. The bar graph represents the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by ANOVA followed by the post hoc test adjusted with a Tukey correction.

Article Snippet: Recombinant mouse Angpt2 proteins (7186-AN), anti-Mouse/Rat Tie2 antibody (AF762), and control polyclonal Goat immunoglobulin G (IgG) (AB-108-C) were purchased from R&D Systems (USA).

Techniques: Inhibition, Transfection, Control, Flow Cytometry, Double Staining, Incubation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Angpt2 Induces Mesangial Cell Apoptosis through the MicroRNA-33-5p-SOCS5 Loop in Diabetic Nephropathy

doi: 10.1016/j.omtn.2018.10.003

Figure Lengend Snippet: Suppressor of Cytokine Signaling 5 Is a Direct Target of miR-33-5p in MMCs (A) The predictive binding score of miR-33-5p and suppressor of cytokine signaling 5 (SOCS5) mRNA according to miRmap database. (B) A schematic representation of sequence alignment of SOCS5 mRNA 3′ UTR based on TargetScan 7.1 version. (C) Luciferase activity was repressed by endogenous miR-33-5p. HEK293 cells were cotransfected with pGL3-SOCS5-3′ UTR luciferase plasmid/pRL-TK Renilla (1:8) or pGL3-SOCS5-3′ UTR MT luciferase plasmid/pRL-TK Renilla (1:8) with various miRNA mimics (control mimic or miR-33-5p mimic) by DharmaFECT Duo transfection reagent after 48 hr, and both firefly and Renilla luciferase activities were quantified using the Dual-Glo Luciferase Assay System. Endogenous SOCS5 in MMCs was regulated by miR-33-5p under an HG condition. (D and E) miR-33-5p inhibitor (D) enhanced and miR-33-5p mimic (E) suppressed SOCS5 expression in MMCs under an NG or HG condition. Cells were transfected with either miR-33-5p inhibitor or miR-33-5p mimic, and, 24 hr after transfection, the cells were treated with Angpt2 for 24 hr under an NG or HG condition. Western blotting was utilized to measure SOCS5 protein expression. (F) Angpt2 increased SOCS5 expression in MMCs under an NG condition, and it augmented SOCS5 expression in MMCs under an HG condition. Western blotting and quantitative analysis of SOCS5 was performed in MMCs treated with Angpt2 under NG or HG for 24 hr. (G and H) The expression of SOCS5 in the MCs of kidneys in mice (G) and humans (H). The kidney sections of C57BL/6 mice, non-diabetic db/m mice, diabetic db/db mice, and human donors (upper tract urothelial carcinoma, UTUC with normal kidney function and normal glomerulus) and patients with DN were co-stained with SOCS5 (brown) and α-SMA (green). The bar graph represents the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by ANOVA followed by the post hoc test adjusted with a Tukey correction.

Article Snippet: Recombinant mouse Angpt2 proteins (7186-AN), anti-Mouse/Rat Tie2 antibody (AF762), and control polyclonal Goat immunoglobulin G (IgG) (AB-108-C) were purchased from R&D Systems (USA).

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Plasmid Preparation, Control, Transfection, Expressing, Western Blot, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Angpt2 Induces Mesangial Cell Apoptosis through the MicroRNA-33-5p-SOCS5 Loop in Diabetic Nephropathy

doi: 10.1016/j.omtn.2018.10.003

Figure Lengend Snippet: Angpt2 Induced MMC Apoptosis through the SOCS5/JAK1/STAT3 Pathway under an HG Condition (A and B) Angpt2 reduced the phosphorylated JAK1:JAK1 ratio (A) and phosphorylated STAT3:STAT3 ratio (B) in MMCs under an NG condition, and it aggravated both reductions in MMCs under an HG condition. Western blotting and quantitative analysis of phosphorylated Janus kinase 1 (JAK1), total JAK1, phosphorylated signal transducer and activator of transcription 3 (STAT3), and total STAT3 were performed in MMCs treated with Angpt2 under an NG or HG condition for 24 hr. (C) Efficacy of SOCS5 siRNA in MMCs. (D) SOCS5 siRNA prevented Angpt2-induced apoptosis in MMCs under an HG condition. Cells were transfected with SOCS5 siRNA, and, 24 hr after transfection, the cells were treated with Angpt2 for 48 hr under an NG or HG condition. Apoptotic cell counts were assessed by flow cytometry with Annexin-V/PI double staining. The bar graph represents the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by Student’s t test or ANOVA followed by the post hoc test adjusted with a Tukey correction.

Article Snippet: Recombinant mouse Angpt2 proteins (7186-AN), anti-Mouse/Rat Tie2 antibody (AF762), and control polyclonal Goat immunoglobulin G (IgG) (AB-108-C) were purchased from R&D Systems (USA).

Techniques: Western Blot, Transfection, Flow Cytometry, Double Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Angpt2 Induces Mesangial Cell Apoptosis through the MicroRNA-33-5p-SOCS5 Loop in Diabetic Nephropathy

doi: 10.1016/j.omtn.2018.10.003

Figure Lengend Snippet: Urinary miR-33-5p Is Negatively Correlated with Renal Dysfunction and Serum Angpt2 in Mice and Humans (A) Urinary miR-33-5p levels were lower in db/db mice (n = 3) compared to db/m mice (n = 3). (B and C) Urinary miR-33-5p was negatively correlated with urinary ACR (B) and serum Angpt2 (C) in mice. (D) Urinary miR-33-5p levels were lower in type 2 DM patients (n = 63) compared to healthy individuals (n = 34). (E and F) Urinary miR-33-5p levels were negatively correlated with urinary ACR (E) and serum Angpt2 (F) in human participants. Extracellular vesicular miR-33-5p in the urine of mice and humans was isolated and then assessed by real-time qPCR. Magnetic Luminex Assay was used to assess serum Angpt2. Urine albumin was measured using an immunoturbidimetric assay, and urine creatinine was determined by the enzymatic method. The bar graph represents the mean ± SEM. *p < 0.05 and **p < 0.01 by Student’s t test or ANOVA followed by the post hoc test adjusted with a Tukey correction, and p value of correlation was analyzed by Spearman analysis.

Article Snippet: Recombinant mouse Angpt2 proteins (7186-AN), anti-Mouse/Rat Tie2 antibody (AF762), and control polyclonal Goat immunoglobulin G (IgG) (AB-108-C) were purchased from R&D Systems (USA).

Techniques: Isolation, Luminex, Immunoturbidimetry Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Angpt2 Induces Mesangial Cell Apoptosis through the MicroRNA-33-5p-SOCS5 Loop in Diabetic Nephropathy

doi: 10.1016/j.omtn.2018.10.003

Figure Lengend Snippet: Illustration of the Mechanism of Angpt2-Induced Apoptosis in MCs through the miR-33-5p-SOCS5 Loop in DN

Article Snippet: Recombinant mouse Angpt2 proteins (7186-AN), anti-Mouse/Rat Tie2 antibody (AF762), and control polyclonal Goat immunoglobulin G (IgG) (AB-108-C) were purchased from R&D Systems (USA).

Techniques: